1. Field of the Invention
This invention relates to the field of flow cytometry and more particularly to the sequential detection of aberrant patterns of protein expression on neoplastic cells and the identification of minimal numbers of neoplastic cells present among a major population of normal cells from blood, bone marrow, spinal fluid and lymph node samples. The invention enables: 1) the unequivocal identification of the aberrant phenotypes carried by neoplastic cells that allow their sensitive and specific identification, 2) the estimation of their utility for the identification of minimal numbers of neoplastic cells displaying an identically aberrant phenotype in another sample from the same individual obtained simultaneously or subsequently, and 3) the calculation of the distribution of neoplastic cells in blood, bone marrow, spinal fluid and lymph node samples both in terms of the percentage of cells and the number of cells per microliter of sample, by means of multiparameter flow cytometry analysis of the cells present in a sample stained with multiple combinations of monoclonal antibodies mixed with a quantified microbead (microparticle) suspension.
2. Background of the Invention
In U.S. Pat. No. 5,047,321, Loken and Terstappen described the multiparameter analysis of cellular components in a body fluid. The body fluids described included blood and bone marrow. Using a combination of two nucleic acid dyes, a fluorescently labeled monoclonal antibody and two light scatter parameters, Loken and Terstappen were able to discriminate among various cellular components of blood and bone marrow, count the number of cells within each component and provide a differential analysis of each of them. Through the combined staining with the LDS-751 (Exciton) DNA-dye, the thiazol orange (TO, Molecular Probes, Inc) RNA-dye and a fluorescently labeled anti-CD45 monoclonal antibody, together with the measurement of forward and sideward light scatter on whole blood and bone marrow aspirates, these authors were able to identify and differentiate between, nucleated red cells, erythrocytes, reticulocytes, platelets, lymphocytes, monocytes, neutrophil granulocytes, basophilic granulocytes, eosinophilic granulocytes and precursors of all nucleated cells. However they could not show the ability of this approach to specifically differentiate between normal and neoplastic cells, coexisting in the same sample.
In U.S. Pat. No. 6,287,791, Terstappen and Chen describe a further refinement of the U.S. Pat. No. 5,047,321, but they did not show any better characterization of the different leukocyte populations.
In U.S. Pat. No. 5,137,809, Loken and Sha describe the multiparameter analysis of cellular components in bone marrow. The authors describe the use, in a first step, of a combination of monoclonal antibodies each labeled with a different fluorochrome, to stain all leukocytes and of further combinations to stain selected populations of leukocytes, in a second step.
All the methods described above were able to identify several populations of normal leukocytes present in blood and bone marrow samples and were only identifying selected subpopulations as identified by the specific combination of monoclonal antibodies and nucleic acid dyes used; nevertheless, they were not able to provide an approach for the specific and reproducible identification of neoplastic cells admixtured naturally or artificially with normal cells in a sample. Also these approaches allow enumeration of the subpopulations of normal leukocytes identified in terms of percentage of total leukocytes. Moreover, by using these methods it is not possible to easily link and directly compare the information on the amount of light scatter and fluorescence measured for cells contained in a first sample to that of cells containing in a second different sample, especially if they derive from different tissues from the same individual, from different individuals or if they have been measured under different conditions.
In U.S. Pat. No. 5,627,037, Ward et al propose a one step technique for the calculation of the number of one or more cell populations contained in a given volume of a blood sample. This approach employs a mixture of reagents containing a mixture of one or more cell markers, a fluorescent quantified microparticle and a fixative. The technique described by Ward et al allows the calculation of the absolute counts of leukocytes, such as CD4+ T-cells, but does not provide any specific indication of the exact procedures to be applied for the enumeration of individual subpopulations of blood leukocytes.
In the last decade many different reports have been published which show that neoplastic cells from a great majority of patients suffering from hematological malignancies display aberrant patterns of antigen expression as detected through the use of several triple and quadruple combinations of monoclonal antibodies analyzed by flow cytometry (Reviewed in Vidriales et al. Best Clin Res Pract, 2003; 16:599-612). These abnormal patterns of antigen expression are never detected in normal cells and they include one or more of the following subtypes: 1) cross-lineage antigen expression, 2) asynchronous antigen expression, 3) antigen over- and under-expression, 4) abnormally high or low light scatter properties, and 5) ectopic phenotypes. Based on these abnormalities several disease-type specific panels of three- and four-color combinations of monoclonal antibody reagents have been proposed for the systematic identification of leukemic cells expressing aberrant phenotypes, in virtually every patient with precursor B-acute lymphoblastic leukemia (BCP-ALL; Lucio et al, Leukemia, 2001; 15: 1185-1192), T-ALL (Porwit-MacDonald, Leukemia, 2000; 14: 816-825), acute myeloblastic leukemia (AML; San Miguel et al, Blood, 2001; 98: 1746-1751), B-cell chronic lymphocytic leukemia (Rawstrom et al, Blood 2001; 98: 29-35) and other B-cell chronic lymphoproliferative disorders (Sanchez et al, Leukemia, 2002; 16: 1460-1469), among other diseases.
In all the approaches described so far for the identification of aberrant phenotypes expressed by neoplastic cells, data interpretation is carried out by an experienced person, with extensive knowledge on the patterns of protein expression on normal cells. Through this approach it has been shown that neoplastic cells from patients studied at first diagnosis and at relapse frequently show more than one aberrant phenotype, especially if relatively large panels of combinations of three or four monoclonal antibodies are used for their identification. Based on all the aberrant phenotypes detected at diagnosis, new 3- or 4-color combinations of monoclonal antibodies for the specific investigation of these neoplastic cell-specific phenotypes are designed and tested for their further use for the investigation of minimal infiltration by neoplastic cells in other samples obtained concurrently (e.g. for staging purposes) or subsequently (e.g. for monitoring the disease and the therapy). However, the need for data interpretation by an expert person with a high amount of knowledge and experience on the patterns of protein expression differentially observed in normal versus neoplastic cells, makes the identification of aberrant phenotypes subjective and difficult to reproduce. Moreover, many of these aberrant phenotypes are only present in a subset of all leukemic cells present in a given sample and they may change in the same patient, and even in another sample from the same tissue, with time. This further makes the identification of aberrant phenotypes, apart from being subjective, uncertain, with potentially occurring false negative and positive results. In addition, current knowledge about the phenotypes of normal cells from blood, bone marrow, spinal fluid and lymph nodes occurring at frequencies of less than 10−4 is very limited; this impacts negatively in the sensitivity of these approaches for detecting minimal numbers of neoplastic cells among a majority of normal blood and bone marrow cells which under the best technical and biological conditions, is currently of between 10−3 (detection of one neoplastic cells among 1000 normal cells) and 10−6 (one neoplastic cell in one million normal cells) depending on the exact lineage, type and maturation stage of the neoplastic cells, the aberrant phenotype used for the identification of the neoplastic cells, and the type of specimen studied.